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Protocol. Genetic transformation of pineapple has been achieved by microprojectile bombardment (biolistics) and by cocultivation with various strains of Agrobacterium tumefaciens (C58, LBA4404, AT2260 and AGL0) and was reviewed by Ko et al. (2008; Table 5.1.1). Genetic transformation is a useful tool to make small, targeted changes to an important cultivar, and it is essential that several cycles of evaluation in the field are done to select those plants that carry the desired transgene in a stable manner (Ko et al., 2013).

Table 5.1.1. Transgenes used for the genetic transformation of pineapple.

Firoozabady and Gutterson (1998) and Firoozabady et al. (2006) cocultivated Agrobacterium with embryogenic cells for 2–3 days in darkness before culturing on a recovery medium without selection pressure for 5–12 days. Sonication and vacuum infiltration were employed to facilitate bacterial penetration into the tissue. Ko et al. (2005), on the other hand, used leaf bases which were cocultivated with Agrobacterium suspensions with 5 min of vacuum infiltration. Cocultivation occurred for 3 weeks under a natural daylight cycle, followed by selection of transformed cells. Cefotaxime is normally used to eliminate excess Agrobacterium following transformation. Espinosa et al. (2002) grew 5-month-old callus tissue in temporary immersion bioreactors (TIBs) to aid selection. Following the initial studies, Yabor et al. (2006) applied the TIB system to study the biochemical side effects that Agrobacterium-mediated pineapple genetic transformation had on the plants. The presence and amounts of compounds related to pathways describing responses to stress and photosynthesis were measured.

Biolistics have also been used to transform pineapple. Nan et al. (1996) reported on transformation efficiency optimization experiments using biolistics and the gus reporter gene on a variety of tissues (parts from crowns, slips and lateral buds of field grown plants); however, the production and evaluation of transformed plants was not reported. Sripaoraya et al. (2001, 2006) targeted leaf bases which were arranged on an agar medium containing mannitol as a pretreatment of the plant tissue 2 h prior to bombardment. Ko et al. (2006) targeted organogenic callus. In these two cases, stable, transformed plants were produced.

To select transformed tissue after cocultivation or bombardment, either antibiotic or herbicide resistance can be incorporated into the vector (Fig. 5.1.2). A stepwise or delayed selection of putative transformed tissue is preferred, to allow recovery from coculture. According to Sripaoraya et al. (2001), there should be no selection pressure for the first 4 weeks after bombardment. Subsequently, cultures should be exposed to 2.8 μM phosphinothricin, which is increased to 5.5 μM 56 days after bombardment. Firoozabady et al. (2006) selected putative transformed cultures with 0.03–0.06 μM chlorsulfuron for 3 weeks after cocultivation, and then increased selection pressure to 0.08–0.14 μM, depending on the type of explant being treated. Ko et al. (2006) selected with 100 μM geneticin for two fortnightly subcultures, followed by 200 μM geneticin with monthly subcultures. Espinosa et al. (2002) and Yabor et al. (2006) cocultivated tissue grown on callus proliferation medium for 4 weeks before selection with 13.8 μM phosphinothricin.

Fig. 5.1.2. Pineapple in vitro leaf bases transformed by cocultivation with Agrobacterium tumefaciens. The plate contains organogenic callus after selection on medium containing geneticin and transformed with nptII and ppo. The callus regenerated morphologically normal plants that were resistant to blackheart (Ko et al., 2013).

Firoozabady et al. (2006) observed that sublethal levels of selection immediately after cocultivation reduced the rate of stable transformation events by 85–95%, and by applying lethal levels of selection immediately after cocultivation or within 20 days of sublethal selection, no transformed tissue was recovered.

Transformation efficiencies of pineapple by either particle bombardment or Agrobacterium-mediated gene delivery are generally c.5%, based on the number of transgenic events per number of explants subjected to the transformation treatment. However, once transgenic cell lines have been obtained, 70–90% of the lines are capable of regenerating whole plants.

ACCOMPLISHMENTS. Several genes have been employed for pineapple transformation (Table 5.1.1). To assess transient and stable transformation for optimizing protocols, the β-glucuronidase (gus or uidA) reporter gene, originating from Escherichia coli, has been widely used (Jefferson et al., 1987). The green fluorescent protein (gfp) gene from Aequorea victoria (jellyfish) has also been used to monitor transformation (Graham et al., 2000; Ko et al., 2006); the use of the gfp gene to monitor transformation is quite convenient as its expression can be monitored in vivo over time in a non-lethal manner, whereas the histochemical gus activity assay is lethal.

The focus has been on economically important transgenes. Successful transformation of pineapple with the bar gene conferring herbicide resistance into ‘Phuket’ was reported by Sripaoraya et al. (2001, 2006). Herbicide resistance improves weed control in the field, thereby reducing competition for available nutrients, resulting in increased fruit yields and economic returns. Furthermore, improved weed control also aids in disease control, as weeds can harbour populations of mealy bugs, ants and aphids, which are known vectors of viruses causing pineapple wilt disease.

Espinosa et al. (2002) and Yabor et al. (2006) introduced two antifungal genes (chi and ap24), to reduce losses caused by Phytophthora nicotianae var. parasitica. The class-I chitinase gene originated from Phaseolus vulgaris, while the ap24 gene was derived from Nicotiana tabacum (Broglie et al., 1986; Woloshuk et al., 1991). These genes promote the degradation of fungus mycelium cell wall components and destabilize fungal membrane potential.

Firoozabady et al. (2006) and Trusov and Botella (2006a) isolated the 1-aminocyclopropane-1-carboxylate (ACC) synthase genes acacs1, acacs2 and acacs3 expressed during pineapple fruit ripening and also involved in flowering. The ACC synthase gene from pineapple is expressed in meristematic cells and activated to induce flowering under certain environmental conditions (low temperatures and photoperiod). Sense and antisense copies of the fruit-related acacs2 and acacs3 transgenes were introduced into pineapple to downregulate the expression of the gene and suppress natural flowering completely. Although pineapple is a non-climacteric fruit, ethylene is still believed to have a significant role during ripening. Considering that harvesting costs take up 34% of the total production costs, synchronized flowering would minimize harvesting and production costs and hence improve the competitiveness of the industry.

Over a 4-year field trial, Trusov and Botella (2006b) drew the following conclusions: (i) constitutive overexpression of an acacs2 gene fragment caused methylation of the endogenous acacs2 gene, resulting in cosuppression of the endogenous acacs2 gene in transgenic plants; and (ii) the transgene alone is not sufficient to cause delayed flowering; however, because of methylation the suppression of the acacs2 gene resulted in down-regulation of gene expression and therefore in significantly delayed flowering.

Another valuable gene which has been successfully targeted in pineapple is polyphenol oxidase (ppo), isolated by Stewart et al. (2001) and described by Ko et al. (2006, 2013; Fig. 5.1.3). Blackheart-resistant pineapple plants have been produced through molecular suppression of ppo activity. Resistance is important as phenolics produced through chilling injury around the core of the fruit (blackheart) are not detectable in intact fruits, resulting in wastage and decreased consumer confidence in the fruit. Blackheart occurs when temperatures in pineapple growing areas in the subtropics are <20°C combined with lower light intensities in the winter months. This can also occur during cold transport of fruit followed by exposure to higher storage temperatures.

Fig. 5.1.3. Transgenic pineapples engineered for blackheart resistance. Note the remnants of bagging of flowers to prevent cross-pollination of non-transformed pineapple sites, a biosecurity requirement of the Australian Office of the Gene Technology Regulator.

Hanke and Flachowsky (2010) commented on pineapple field trials that were focused on improved resistance to nematodes and viral diseases. Other trials were focused on plants with altered agronomic properties and improved product quality. Del Monte has obtained red-fleshed ‘Rosé’ pineapple by combining overexpression of a gene derived from tangerine and suppression of other genes to increase the accumulation of lycopene (Ogata et al., 2016).

In the future, combining traits, e.g. improved fruit quality, control of natural flowering and/or pathogen resistance with herbicide resistance, is likely to be an effective goal, thereby facilitating more effective weed control without damaging the crop and introducing a combination of desirable traits.

While transformation in the age of genomics offers enormous potential for pineapple improvement, public concerns about genetically modified crops with respect to health, environmental and ownership issues are currently hindering acceptance of transgenic products in many countries and are issues that need to be addressed.